Small molecules, enormous functions: potential approach for overcoming bottlenecks in embryogenic tissue induction and maintenance in conifers

Abstract

Somatic embryogenesis (SE) is not only the most effective method among various strategies for the asexual propagation of forest trees but also a basis for genetic improvement. However, some bottlenecks, such as the recalcitrance of initiation, the maintenance of embryogenic potential during proliferation and the low efficiency of maturation as well as high rate of abnormal embryo development remain unresolved. These bottlenecks refer to complex mechanisms, including transcriptional regulatory networks, epigenetic modifications and physiological conditions. In recent years, several small molecules utilized in animal stem cell research have exhibited positive effects on plant regeneration, including conifer species, which offers a potential novel approach to overcome the challenges associated with SE in conifers. In this review, we summarize the small molecules used in conifers, including redox substances, epigenetic regulatory inhibitors and other metabolism-related molecules, which overcome these difficulties without the use of genetic engineering. Moreover, this approach also has the advantages of dynamic reversibility, simple operation, and simultaneous regulation of multiple targets, which might be one of the best choices for optimizing plant regeneration systems including SE.

Acknowledgements

This paper was supported by the National Key R&D Program of China (2023YFD2200104), the National Natural Science Foundation of China (32271836), and the Fundamental Research Funds for the Central Universities (2019ZY39).

Author contributions

T.G. and J.Z. designed the review plan. T.G., F.B., and Y.M.F. searched the references. T.G. and F.B. wrote the manuscript. J.F.Z. and J.Z. were involved in manuscript editing. All the authors have read and approved the final manuscript.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

References

[1] Park YS. Implementation of conifer somatic embryogenesis in clonal forestry: technical requirements and deployment considerations. Ann For Sci. 2002; 59 :651-6

[2] Lelu-Walter MA, Thompson D, Harvengt L. et al. Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genet Genomes. 2013; 9 :883-99

[3] Zhu T, Wang J, Hu J. et al. Mini review: application of the somatic embryogenesis technique in conifer species. Forestry Research. 2022; 2, 18 :

[4] Sun C, Xie YH, Li Z. et al. The Larix kaempferi genome reveals new insights into wood properties. J Integr Plant Biol. 2022; 64 : 1364-73

[5] Niu S, Li J, Bo W. et al. The Chinese pine genome and methylome unveil key features of conifer evolution. Cell. 2022; 185 : 204-217.e14

[6] Poovaiah C, Phillips L, Geddes B. et al. Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don). BMC Plant Biol. 2021; 21 :363

[7] Cui Y, Zhao J, Gao Y. et al. Efficient multi-sites genome editing and plant regeneration via 751891 somatic embryogenesis in Picea glauca. Front Plant Sci. 2021; 12 :751891

[8] Hassani SB, Trontin J, Raschke J. et al. Constitutive overexpression of a conifer WOX2 homolog affects somatic embryo development in Pinus pinaster and promotes somatic embryogenesis and organogenesis in Arabidopsis seedlings. Front Plant Sci. 2022; 13 :838421

[9] Castander-Olarieta A, Montalbán IA, Moncaleán P. Multi-strategy approach towards optimization of maturation and germination in radiata pine somatic embryogenesis. Plant Cell, Tissue and Organ Culture (PCTOC). 2023; 153 :173-90

[10] Pullman GS, Johnson S, Peter G. et al. Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination, and gene expression. Plant Cell Rep. 2003; 21 :747-58

[11] Varis S, Klimaszewska K, Aronen T. Somatic embryogenesis and plant regeneration from primordial shoot explants of Picea abies (L.) H. Karst. Somatic trees. Front Plant Sci. 2018; 9 :1551

[12] Salaj T, Fráterová L, Cárach M. et al. The effect of culture medium formulation on Pinus nigra somatic embryogenesis. Dendrobiology. 2013; 119-128 :119-28

[13] Walther M, Wagner I, Raschke J. et al. Abscisic acid induces somatic embryogenesis and enables the capture of high-value genotypes in Douglas fir (Pseudotsuga menziesii [MIRB.] Franco). Plant Cell, Tissue and Organ Culture (PCTOC). 2022; 148 :45-59

[14] Horstman A, Bemer M, Boutilier K. A transcriptional view on somatic embryogenesis. Regeneration. 2017; 4 :201-16

[15] Bouchabke-Coussa O, Obellianne M, Linderme D. et al. Wuschel overexpression promotes somatic embryogenesis and induces organogenesis in cotton (Gossypium hirsutum L.) tissues cultured in vitro. Plant Cell Rep. 2013; 32 :675-86

[16] Valencia-Lozano E, Cabrera-Ponce JL, Barraza A. et al. Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-tom. PLoS One. 2024; 19 :e0301169

[17] De La Torre AR. et al. Functional and morphological evolution in gymnosperms: A portrait of implicated gene families. Evol Appl. 2020; 13 :210-27

[18] Ranade SS, Egertsdotter U. In silico characterization of putative gene homologues involved in somatic embryogenesis suggests that some conifer species may lack LEC2, one of the key regulators of initiation of the process. BMC Genomics. 2021; 22 :392

[19] Wang J, Sun S, Deng H. Chemical reprogramming for cell fate manipulation: methods, applications, and perspectives. Cell Stem Cell. 2023; 30 :1130-47

[20] Valero-Rubira I, Vallés MP, Echávarri B. et al. New epigenetic modifier inhibitors enhance microspore embryogenesis in bread wheat. Plan Theory. 2024; 13 :772

[21] Hicks GR, Raikhel NV. Small molecules present large opportunities in plant biology. Annu Rev Plant Biol. 2012; 63 :261-82

[22] Berenguer E, Carneros E, Pérez-Pérez Y. et al. Small molecule inhibitors of mammalian GSK-3beta promote in vitro plant cell reprogramming and somatic embryogenesis in crop and forest species. J Exp Bot. 2021; 72 :7808-25

[23] Wu H, Zhang K, Li J. et al. Somatic embryogenesis from mature sorghum seeds: an underutilized genome editing recipient system. Heliyon. 2024; 10 :e23638

[24] Zhu J, Zhang K, Xiong H. et al. H2O2 significantly affects Larix kaempferi × Larix olgensis somatic embryogenesis. Int J Mol Sci. 2024; 25 :669

[25] Zhou T, Yang X, Guo K. et al. ROS homeostasis regulates somatic embryogenesis via the regulation of auxin signaling in cotton. Mol Cell Proteomics. 2016; 15 :2108-24

[26] Calabuig-Serna A, Mir R, Arjona P. et al. Calcium dynamics and modulation in carrot somatic embryogenesis. Front Plant Sci. 2023; 14 :1150198

[27] Wang Y, Yao R. H2O2 and Ca2+ are involved in the embryogenic potential loss of cells during long-term proliferation culture in Pinus massoniana. Plant Cell, Tissue and Organ Culture (PCTOC). 2023; 154 :657-72

[28] Vondrakova Z, Dobrev PI, Pesek B. et al. Profiles of endogenous Phytohormones over the course of Norway spruce somatic embryogenesis. Front Plant Sci. 2018; 9 :1283

[29] Zhou X, Zheng R, Liu G. et al. Desiccation treatment and endogenous IAA levels are key factors influencing high frequency somatic embryogenesis in Cunninghamia lanceolata (lamb.) hook. Front Plant Sci. 2017; 8 :2054

[30] Fehér A. Somatic embryogenesis — stress-induced remodeling of plant cell fate. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 2015; 1849 :385-402

[31] Méndez-Hernández HA, Ledezma-Rodríguez M, Avilez-Montalvo RN. et al. Signaling overview of plant somatic embryogenesis. Front Plant Sci. 2019; 10 :77

[32] Elbl PM, de Souza DT, Rosado D. et al. Building an embryo: an auxin gene toolkit for zygotic and somatic embryogenesis in Brazilian pine. Gene. 2022; 817 :146168

[33] Maruyama TE, Ueno S, Hosoi Y. et al. Somatic embryogenesis initiation in Sugi (Japanese cedar, Cryptomeria japonica D. Don): responses from male-fertile, male-sterile, and Polycross-pollinated-derived seed explants. Plan Theory. 2021; 10 :398

[34] Hargreaves CL, Reeves CB, Find KI. et al. Overcoming the challenges of family and genotype representation and early cell line proliferation in somatic embryogenesis from control-pollinated seeds of Pinus radiata. N Z J For Sci. 2011; 41 : 97-114

[35] Niskanen AM, Lu J, Seitz S. et al. Effect of parent genotype on somatic embryogenesis in scots pine (Pinus sylvestris ). Tree Physiol. 2004; 24 :1259-65

[36] MacKay JJ, Becwar MR, Park YS. et al. Genetic control of somatic embryogenesis initiation in loblolly pine and implications for breeding. Tree Genet Genomes. 2006; 2 :1-9

[37] Carneros E, Celestino C, Klimaszewska K. et al. Plant regeneration in stone pine (Pinus pinea L.) by somatic embryogenesis. Plant Cell, Tissue and Organ Culture (PCTOC). 2009; 98 :165-78

[38] Hosoi Y, Maruyama TE. Plant regeneration from embryogenic tissue of Pinus luchuensis Mayr, an endemic species in Ryukyu Island, Japan. Plant Biotechnology. 2012; 29 :401-6

[39] Hazubska-Przybył T, Ratajczak E, Obarska A. et al. Different roles of auxins in somatic embryogenesis efficiency in two Picea species. Int J Mol Sci. 2020; 21 :3394

[40] Gao F, Cao X, Qin C. et al. Effects of plant growth regulators and sucrose on proliferation and quality of embryogenic tissue in Picea pungens. Sci Rep. 2023; 13 :13194

[41] Pullman GS, Zeng X, Copeland-Kamp B. et al. Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents. Tree Physiol. 2015; 35 :209-24

[42] Trontin J. et al. An overview of current achievements and shortcomings in developing maritime pine somatic embryogenesis and enabling technologies in France. In Proceedings of the 1st IUFRO 2 September 2002 Conference, Advances in Somatic Embryogenesis of Trees and Its Application for the Future Forests and Plantations, Suwon, Korea, 19-21 August 2010. 2011

[43] Montalbán IA, De Diego N, Moncaleán P. Enhancing initiation and proliferation in radiata pine (Pinus radiata D. Don) somatic embryogenesis through seed family screening, zygotic embryo staging and media adjustments. Acta Physiol Plant. 2012; 34 : 451-60

[44] Pullman GS, Bucalo K. Pine somatic embryogenesis using zygotic embryos as explants. Methods Mol Biol. 2011; 710 :267-91

[45] Kim YW. Initiation of embryogenic callus from mature zygotic embryos in Japanese larch (Larix kaempferi ). J Plant Biotechnol. 2015; 42 :223-7

[46] Salaj T, Klubicová K, Matusova R. et al. Somatic embryogenesis in selected conifer trees Pinus nigra Arn. and Abies hybrids. Front Plant Sci. 2019; 10 :13

[47] Hu R, Sun Y, Wu B. et al. Somatic embryogenesis of immature Cunninghamia lanceolata (lamb.) hook zygotic embryos. Sci Rep. 2017; 7 :56

[48] Bonga JM, Klimaszewska KK, von Aderkas P. Recalcitrance in clonal propagation, in particular of conifers. Plant Cell, Tissue and Organ Culture (PCTOC). 2010; 100 :241-54

[49] Lara-Chavez A, Flinn BS, Egertsdotter U. Initiation of somatic embryogenesis from immature zygotic embryos of Oocarpa pine (Pinus oocarpa Schiede ex Schlectendal). Tree Physiol. 2011; 31 :539-54

[50] Wang F, Shang G, Wang J. Towards a hierarchical gene regulatory network underlying somatic embryogenesis. Trends Plant Sci. 2022; 27 :1209-17

[51] Zhao R, Qi S, Cui Y. et al. Transcriptomic and physiological analysis identifies a gene network module highly associated with brassinosteroid regulation in hybrid sweetgum tissues differing in the capability of somatic embryogenesis. Hort Res. 2022; 9 :uhab047

[52] Bashir MA, Silvestri C, Salimonti A. et al. Can ethylene inhibitors enhance the success of olive somatic embryogenesis? Plan Theory. 2022; 11 :168

[53] Abdelsalam A, Mahran E, Chowdhury K. et al. Effect of exogenous methyl jasmonate on in vitro propagation, metabolic profiling and proximadiol production from Cymbopogon schoenanthus subsp. proximus. Indian J Plant Physiol. 2021; 26 : 548-60

[54] Zhou LQ, Dean J. Reprogramming the genome to totipotency in mouse embryos. Trends Cell Biol. 2015; 25 :82-91

[55] Osorio-Montalvo P, Sáenz-Carbonell L, De-la-Peña C. 5-Azacytidine: A promoter of epigenetic changes in the quest to improve plant somatic embryogenesis. Int J Mol Sci. 2018; 19 : 3182

[56] Karami O, Rahimi A, Mak P. et al. An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication. Nat Commun. 2021; 12 : 2508

[57] Abrahamsson M, Valladares S, Merino I. et al. Degeneration pattern in somatic embryos of Pinus sylvestris L. In Vitro Cell Dev Biol - Plant. 2017; 53 :86-96

[58] Breton D, Harvengt L, Trontin J. et al. Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine. Plant Cell Tissue Organ Cult. 2006; 87 :95-108

[59] Klimaszewska K, Noceda C, Pelletier G. et al. Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.). In Vitro Cell Dev Biol - Plant. 2009; 45 :20-33§

[60] Breton D, Harvengt L, Trontin J. et al. High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine. In Vitro Cell Dev Biol - Plant. 2005; 41 :494-504

[61] Gao F, Peng C, Wang H. et al. Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis. J For Res. 2021; 32 :483-91

[62] Pullman GS. Embryogenic tissue initiation in Loblolly Pine (Pinus Taeda L.). In: Jain SM, Gupta P (eds.), Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants, Vol. 1. Cham:Springer International Publishing, 2018, 13-31

[63] Pullman GS, Olson K, Fischer T. et al. Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation. New For. 2016; 47 : 453-80

[64] Stasolla C, Yeung EC. Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca ) somatic embryos. Plant Physiol Biochem. 2007; 45 :188-98

[65] Abrahamsson M, Silvia B, Bullet V. et al. Patterning during somatic embryogenesis in scots pine in relation to polar auxin transport and programmed cell death. Plant Cell Tissue Org Cult. 2012; 109 :391-400

[66] Klimaszewska K, Hargreaves C, Lelu-Walter M. et al. Advances in conifer somatic embryogenesis since year Germana MA, Lambardi M (eds.), Vitro Embryogenesis in Higher Plants, Vol. 1359. New York, NY: Humana Press, 2015, 131-66

[67] Jiang S, Chen X, Gao Y. et al. Plant regeneration via somatic embryogenesis in Larix principis-rupprechtii Mayr. Forests. 2021; 12 :1335

[68] Krajňáková J, Häggman H. Somatic Embryogenesis of Abies cephalonica Loud. In Vitro Embryogenesis in Higher Plants, Vol. 1359. New York, NY: Humana Press, 2015, 417-30

[69] Pérez-Oliver MA, Haro JG, Pavlović I. et al. Priming maritime pine Megagametophytes during somatic embryogenesis improved plant adaptation to heat stress. Plan Theory. 2021; 10 :446

[70] Find J, Grace L, Krogstrup P. Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana ). Physiol Plant. 2002; 116 :231-7

[71] Maruyama TE, Hosoi Y. Protocol for Somatic Embryogenesis in Japanese Black Pine (Pinus thunbergii Parl.) and Japanese Red Pine (Pinus densiflora Sieb. et Zucc.). In: Jain SM, Gupta P,eds. (eds), Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants. Cham: Springer International Publishing, 2018, 229-41

[72] Maruyama TE, Hosoi Y. Progress in somatic embryogenesis of Japanese pines. Front Plant Sci. 2019; 10 :031

[73] Li Q, Deng C, Zhu T. et al. Dynamics of physiological and miRNA changes after long-term proliferation in somatic embryogenesis of Picea balfouriana. Trees. 2019; 33 :469-80

[74] Konagaya KI, Nanasato Y, Taniguchi T. A protocol for agrobacterium-mediated transformation of Japanese cedar, Sugi (Cryptomeria japonica D. Don) using embryogenic tissue explants. Plant Biotechnol (Tokyo). 2020; 37 :147-56

[75] Välimäki S, Teyssier C, Tikkinen M. et al. Norway spruce somatic embryogenesis benefits from proliferation of embryogenic tissues on filter discs and cold storage of cotyledonary embryos. Front Plant Sci. 2022; 13 :1031686

[76] Wang J, Zhang L, Qi L. et al. Integrated transcriptomic and metabolic analyses provide insights into the maintenance of embryogenic potential and the biosynthesis of phenolic acids and flavonoids involving transcription factors in Larix kaempferi (lamb.) Carr. Front Plant Sci. 2022; 13 :1056930

[77] Peng C, Gao F, Wang H. et al. Morphological and physiological indicators for screening cell lines with high potential for somatic embryo maturation at an early stage of somatic embryogenesis in Pinus koraiensis. Plan Theory. 2022; 11 :1867

[78] Avilez-Montalvo JR, Quintana-Escobar AO, Méndez-Hernández HA. et al. Auxin-Cytokinin cross talk in somatic embryogenesis of Coffea canephora. Plan Theory. 2022; 11 :2013

[79] Karami O, de Jong H, Somovilla VJ. et al. Structure-activity relationship of 2,4-D correlates auxinic activity with the induction of somatic embryogenesis in Arabidopsis thaliana. Plant J. 2023; 116 :1355-69

[80] El Meskaoui A, Tremblay FM. Involvement of ethylene in the maturation of black spruce embryogenic cell lines with different maturation capacities. J Exp Bot. 2001; 52 :761-9

[81] von Aderkas P, Bonga JM. Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment. Tree Physiol. 2000; 20 :921-8

[82] Pattyn J, Vaughan Hirsch J, Van de Poel B. The regulation of ethylene biosynthesis: a complex multilevel control circuitry. New Phytol. 2021; 229 :770-82

[83] Mori IC, Schroeder JI. Reactive oxygen species activation of plant Ca2+ channels. A signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol. 2004; 135 :702-8

[84] Liang Y, Xu X, Shen H. et al. Morphological and endogenous phytohormone changes during long-term embryogenic cultures in Korean pine. Plant Cell, Tissue and Organ Culture (PCTOC). 2022; 151 :253-64

[85] Vondráková Z, Eliášová K, Fischerová L. et al. The role of auxins in somatic embryogenesis of Abies alba. Open Life Sciences. 2011; 6 :587-96

[86] Karami O, Philipsen C, Rahimi A. et al. Endogenous auxin maintains embryonic cell identity and promotes somatic embryo development in Arabidopsis. Plant J. 2023; 113 :7-22

[87] Fraga HPF, Vieira LN, Heringer AS. et al. DNA methylation and proteome profiles of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures as affected by plant growth regulators supplementation. Plant Cell, Tissue and Organ Culture (PCTOC). 2016; 125 :353-74

[88] Chen X, Xu X, Shen X. et al. Genome-wide investigation of DNA methylation dynamics reveals a critical role of DNA demethylation during the early somatic embryogenesis of Dimocarpus longan Lour. Tree Physiol. 2020; 40 :1807-26

[89] Rival A, Ilbert P, Labeyrie A. et al. Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures. Plant Cell Rep. 2013; 32 : 359-68

[90] Ji L, Mathioni SM, Johnson S. et al. Genome-wide reinforcement of DNA methylation occurs during somatic embryogenesis in soybean. Plant Cell. 2019; 31 :2315-31

[91] Aronen T, Virta S, Varis S. Telomere length in Norway spruce during somatic embryogenesis and cryopreservation. Plan Theory. 2021; 10 :416

[92] Harry IS, Thorpe TA. Somatic embryogenesis and plant regeneration from mature zygotic embryos of red spruce. Bot Gaz. 1991; 152 :446-52

[93] Ozudogru EA, Lambardi M. Cryotechniques for the long-term conservation of embryogenic cultures from woody plants. Methods Mol Biol. 2016; 1359 :537-50

[94] Hazubska-Przybył T, Wawrzyniak MK, Obarska A. et al. Cryopreservation of Abies alba × A. numidica and Pinus nigra embryogenic tissues by stepwise dehydration method. Plant Methods. 2024; 20 :10

[95] Nakamura M, Batista RA, Köhler C. et al. Polycomb repressive complex 2-mediated histone modification H3K27me3 is associated with embryogenic potential in Norway spruce. J Exp Bot. 2020; 71 :6366-78

[96] Considine MJ, Foyer CH. Oxygen and reactive oxygen species-dependent regulation of plant growth and development. Plant Physiol. 2021; 186 :79-92

[97] Considine MJ, Foyer CH. Stress effects on the reactive oxygen species-dependent regulation of plant growth and development. J Exp Bot. 2021; 72 :5795-806

[98] Belmonte MF, Donald G, Reid DM. et al. Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca ). J Exp Bot. 2005; 56 :2355-64

[99] Stasolla C. Glutathione redox regulation of in vitro embryogenesis. Plant Physiol Biochem. 2010; 48 :319-27

[100] Belmonte MF, Ambrose SJ, Ross ARS. et al. Improved development of microspore-derived embryo cultures of Brassica napus cv topaz following changes in glutathione metabolism. Physiol Plant. 2006; 127 :690-700

[101] Izuno A, Maruyama TE, Ueno S. et al. Genotype and transcriptome effects on somatic embryogenesis in Cryptomeria japonica. PLoS One. 2020; 15 :e0244634

[102] Gao F, Wang R, Shi Y. et al. Reactive oxygen metabolism in the proliferation of Korean pine embryogenic callus cells promoted by exogenous GSH. Sci Rep. 2023; 13 :1-12

[103] Gao F, Peng C, Zhang Y. et al. Glutathione plays a positive role in the proliferation of Pinus koraiensis embryogenic cells. Int J Mol Sci. 2022; 23 :14679

[104] Peng CX, Gao F, Wang H. et al. Physiological and biochemical traits in Korean pine somatic embryogenesis. FORESTS. 2020; 11 :

[105] Belmonte MF, Stasolla C, Katahira R. et al. Glutathione-induced growth of embryogenic tissue of white spruce correlates with changes in pyrimidine nucleotide metabolism. Plant Sci. 2005; 168 :803-12

[106] Belmonte MF, Stasolla C. Applications of dl-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca ) somatic embryo development. Plant Cell Rep. 2007; 26 :517-23

[107] Belmonte MF, Donald G, Reid DM. et al. Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca ). J Exp Bot. 2005; 56 :2355-64

[108] Efroni I. A conceptual framework for cell identity transitions in plants. Plant Cell Physiol. 2018; 59 :696-706

[109] Liu X, Zhu K, Xiao J. Recent advances in understanding of the epigenetic regulation of plant regeneration. aBIOTECH. 2023; 4 : 31-46

[110] Peng J, Zhang WJ, Zhang Q. et al. The dynamics of chromatin states mediated by epigenetic modifications during somatic cell reprogramming. Frontiers Cell Dev Biol. 2023; 11 : 1097780

[111] Grzybkowska D, Morończyk J, Wójcikowska B. et al. Azacitidine (5-AzaC)-treatment and mutations in DNA methylase genes affect embryogenic response and expression of the genes that are involved in somatic embryogenesis in Arabidopsis. Plant Growth Regul. 2018; 85 :243-56

[112] Solís M, El-Tantawy A, Cano V. et al. 5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation, but prevents subsequent embryo development in rapeseed and barley. Front Plant Sci. 2015; 6 :472

[113] Nic-Can GI, López-Torres A, Barredo-Pool F. et al. New insights into somatic embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 are epigenetically regulated in Coffea canephora. PLoS One. 2013; 8 :e72160

[114] Osorio-Montalvo P, de-la-Peña C, Oropeza C. et al. A peak in global DNA methylation is a key step to initiate the somatic embryogenesis of coconut palm (Cocos nucifera L). Plant Cell Rep. 2020; 39 :1345-57

[115] Leljak-Levanic D, Bauer N, Mihaljevic S. et al. Changes in DNA methylation during somatic embryogenesis in Cucurbita pepo L. Plant Cell Rep. 2004; 23 :120-7

[116] Santos D, Fevereiro P. Loss of DNA methylation affects somatic embryogenesis in Medicago truncatula. Plant Cell Tissue Organ Cult. 2002; 70 :155-61

[117] Leljak-Levanic D, Mihaljevic S, Jelaska S. Variations IN DNA methylation IN Piceo omorika (Panc) purk. Embryogenic tissue and the ability for embryo maturation. Propogation Ornamental Plants. 2009; 9 :3-9

[118] Gao Y, Chen X, Liu C. et al. Involvement of 5mC DNA demethylation via 5-aza-2′-deoxycytidine in regulating gene expression during early somatic embryo development in white spruce (Picea glauca). Forestry Research. 2023; 3 :30

[119] Pila Quinga LA, De Freitas P, Fraga H. et al. Epigenetics of long-term somatic embryogenesis in Theobroma cacao L.: DNA methylation and recovery of embryogenic potential. Plant Cell, Tissue and Organ Culture (PCTOC). 2017; 131 :295-305

[120] Berenguer E, Bárány I, Solís MT. et al. Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore Totipotency and enhances embryogenesis initiation. Front Plant Sci. 2017; 8 :1161

[121] Wójcikowska B, Botor M, Morończyk J. et al. Trichostatin A triggers an embryogenic transition in Arabidopsis explants via an auxin-related pathway. Front Plant Sci. 2018; 9 :1353

[122] Morończyk J, Bra˛szewska A, Wójcikowska B. et al. Insights into the histone acetylation-mediated regulation of the transcription factor genes that control the embryogenic transition in the somatic cells of Arabidopsis. Cells. 2022; 11 :863

[123] Iwase A, Takebayashi A, Aoi Y. et al. 4-Phenylbutyric acid promotes plant regeneration as an auxin by being converted to phenylacetic acid via an IBR3-independent pathway. Plant Biotechnology. 2022; 39 :51-8

[124] Zhang X, Lai C, Xu L. et al. Integrated proteome and acetylome analyses provide novel insights into early somatic embryogenesis of Dimocarpus longan. Plant Physiol Biochem. 2023; 196 :903-16

[125] Nowak K, Wójcikowska B, Gajecka M. et al. The improvement of the in vitro plant regeneration in barley with the epigenetic modifier of histone acetylation, trichostatin A. J Appl Genet. 2024; 65 :13-30

[126] Uddenberg D, Valladares S, Abrahamsson M. et al. Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor. Planta. 2011; 234 :527-39

[127] Martinez O, Arjones V, Gonzalez MV. et al. Histone deacetylase inhibitors increase the embryogenic potential and alter the expression of embryogenesis-related and HDAC-encoding genes in grapevine (Vitis vinifera L., cv. Mencia). Plants (Basel). 2021; 10 :1164

[128] Bie XM, Dong L, Li XH. et al. Trichostatin A and sodium butyrate promotes plant regeneration in common wheat. Plant Signal Behav. 2020; 15 :1820681

[129] Kumar V, Thakur JK, Prasad M. Histone acetylation dynamics regulating plant development and stress responses. Cell Mol Life Sci. 2021; 78 :4467-86

[130] Nguyen CT, Tran G, Nguyen NH. Homeostasis of histone acetylation is critical for auxin signaling and root morphogenesis. Plant Mol Biol. 2020; 103 :1-7

[131] Kim JY, Yang W, Forner J. et al. Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis. EMBO J. 2018; 37 :e98726

[132] Temman H, Sakamoto T, Ueda M. et al. Histone deacetylation regulates de novo shoot regeneration. PNAS Nexus. 2023; 2 :pgad002

[133] Valero-Rubira I, Castillo AM, Burrell MÁ. et al. Microspore embryogenesis induction by mannitol and TSA results in a complex regulation of epigenetic dynamics and gene expression in bread wheat. Front Plant Sci. 2023; 13 :1058421

[134] Wójcikowska B, Wójcik AM, Gaj MD. Epigenetic regulation of auxin-induced somatic embryogenesis in plants. Int J Mol Sci. 2020; 21 :2307

[135] He C, Chen X, Huang H. et al. Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS Genet. 2012; 8 :e1002911

[136] Zhu T, Hu J, Yang X. et al. Analysis of polycomb repressive complex 2 (PRC2) subunits in Picea abies with a focus on embryo development. BMC Plant Biol. 2023; 23 :347

[137] Liu C, Lu F, Cui X. et al. Histone methylation in higher plants. Annu Rev Plant Biol. 2010; 61 :395-420

[138] Zhang H, Lang Z, Zhu J. Dynamics and function of DNA methylation in plants. Nat Rev Mol Cell Biol. 2018; 19 :489-506

[139] Ausin I, Feng S, Yu C. et al. DNA methylome of the 20-gigabase Norway spruce genome. Proc Natl Acad Sci. 2016; 113 :E8106-13

[140] Bravo S, Bertín A, Turner A. et al. Differences in DNA methylation, DNA structure and embryogenesis-related gene expression between embryogenic and non embryogenic lines of Pinus radiata D. Don. Plant Cell, Tissue and Organ Culture (PCTOC). 2017; 130 :521-9

[141] Noceda C, Salaj T, Pérez M. et al. DNA demethylation and decrease on free polyamines is associated with the embryogenic capacity of Pinus nigra Arn. cell culture. Trees. 2009; 23 : 1285-93

[142] Gao Y, Cui Y, Zhao R. et al. Cryo-treatment enhances the Embryogenicity of mature somatic embryos via the lncRNA-miRNA-mRNA network in white spruce. Int J Mol Sci. 2022; 23 :1111

[143] Castander-Olarieta A, Pereira C, Sales E. et al. Induction of Radiata pine somatic embryogenesis at high temperatures provokes a long-term decrease in DNA methylation/hydroxymethylation and differential expression of stress-related genes. Plan Theory. 2020; 9 :1762

[144] Pereira C, Castander-Olarieta A, Sales E. et al. Heat stress in Pinus halepensis somatic embryogenesis induction: effect in DNA methylation and differential expression of stress-related genes. Plan Theory. 2021; 10 :2333

[145] Teyssier C, Maury S, Beaufour M. et al. In search of markers for somatic embryo maturation in hybrid larch (Larix x eurolepis): global DNA methylation and proteomic analyses. Physiol Plant. 2014; 150 :271-91

[146] Grzybkowska D, Nowak K, Gaj MD. Hypermethylation of auxin-responsive motifs in the promoters of the transcription factor genes accompanies the somatic embryogenesis induction in Arabidopsis. Int J Mol Sci. 2020; 21 :6849

[147] Bown AW, Shelp BJ. Plant GABA: not just a metabolite. Trends Plant Sci. 2016; 21 :811-3

[148] Pei L, Zhao Y, Shi X. et al. The role of γ-aminobutyric acid (GABA) in the occurrence of adventitious roots and somatic embryos in woody plants. Plan Theory. 2022; 11 :3512

[149] Booz MR, Kerbauy GB, Guerra MP. et al. The role of γ-aminobutyric acid (Gaba) in somatic embryogenesis of Acca sellowiana Berg. (Myrtaceae). Braz J Plant Physiol. 2009; 21 :271-80

[150] Chen T, Yang D, Fan R. et al. γ-Aminobutyric acid a novel candidate for rapid induction in somatic embryogenesis of liriodendron hybrid. Plant Growth Regul. 2022; 96 :293-302

[151] Dowlatabadi R, Weljie AM, Thorpe TA. et al. Metabolic footprinting study of white spruce somatic embryogenesis using NMR spectroscopy. Plant Physiol Biochem. 2009; 47 :343-50

[152] De Diego N, Saiz-Fernandez I, Rodriguez JL. et al. Metabolites and hormones are involved in the intraspecific variability of drought hardening in radiata pine. J Plant Physiol. 2015; 188 : 64-71

[153] Zhang H, Hu Z, Lei C. et al. A plant Phytosulfokine peptide initiates auxin-dependent immunity through cytosolic Ca(2+) signaling in tomato. Plant Cell. 2018; 30 :652-67

[154] Ding S, Lv J, Hu Z. et al. Phytosulfokine peptide optimizes plant growth and defense via glutamine synthetase GS2 phosphorylation in tomato. EMBO J. 2023; 42 :e111858

[155] Wang J, Li H, Han Z. et al. Allosteric receptor activation by the plant peptide hormone phytosulfokine. Nature. 2015; 525 : 265-8

[156] Hanai HNUJ, Matsuno T, Yamamoto M. et al. A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot [Daucus carota ] somatic embryo formation. Plant Cell Physiol. 2000; 41 :27-32

[157] Ochatt S, Conreux C, Moussa Mcolo R. et al. Phytosulfokine-alpha, an enhancer of in vitro regeneration competence in recalcitrant legumes. Plant Cell, Tissue and Organ Culture (PCTOC). 2018; 135 :189-201

[158] Umehara M, Ogita S, Sasamoto H. et al. Two stimulatory effects of the peptidyl growth factor phytosulfokine during somatic embryogenesis in Japanese larch (Larix leptolepis Gordon). Plant Sci. 2005; 169 :901-7

[159] Igasaki T, Akashi N, Ujino-Ihara T. et al. Phytosulfokine stimulates somatic embryogenesis in Cryptomeria japonica. Plant Cell Physiol. 2003; 44 :1412-6

[160] Hao Z, Wu H, Zheng R. et al. The plant peptide hormone phytosulfokine promotes somatic embryogenesis by maintaining redox homeostasis in Cunninghamia lanceolata. Plant J. 2023; 113 : 716-33

[161] Asif M, Eudes F, Randhawa H. et al. Phytosulfokine alpha enhances microspore embryogenesis in both triticale and wheat. Plant Cell, Tissue and Organ Culture (PCTOC). 2014; 116 : 125-30

[162] Wu H, Zheng R, Hao Z. et al. Cunninghamia lanceolata PSK peptide hormone genes promote primary root growth and adventitious root formation. Plants (Basel). 2019; 8 :520

[163] Mestinšek Mubič S, Kunej U, Vogrinčič V. et al. The effect of phytosulfokine alpha on haploid embryogenesis and gene expression of Brassica napus microspore cultures. Front Plant Sci. 2024; 15 :1336519

[164] Yoo M, Albert VA, Soltis PS. et al. Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants. BMC Plant Biol. 2006; 6 :3

[165] Nie S, Yan Y, Wang Y. et al. Proper doses of brassinolide enhance somatic embryogenesis in different competent Korean pine cell lines during embryogenic callus differentiation. Front Plant Sci. 2024; 15 :1330103

[166] Lee HG, Jang SY, Jie EY. et al. Adenosine monophosphate enhances callus regeneration competence for de novo plant organogenesis. Mol Plant. 2023; 16 :1867-70

[167] Murvanidze N, Nisler J, Leroux O. et al. Cytokinin oxidase/dehydrogenase inhibitors stimulate 2iP to induce direct somatic embryogenesis in Coffea arabica. Plant Growth Regul. 2021; 94 : 195-200

[168] Duchow S, Dahlke RI, Geske T. et al. Arabinogalactan-proteins stimulate somatic embryogenesis and plant propagation of pelargonium sidoides. Carbohydr Polym. 2016; 152 : 149-55

[169] Wang F, Shang GD, Wu LY. et al. Chromatin accessibility dynamics and a hierarchical transcriptional regulatory network structure for plant somatic embryogenesis. Dev Cell. 2020; 54 : 742-757.e8

[170] Zhao L, Yang Y, Chen J. et al. Dynamic chromatin regulatory programs during embryogenesis of hexaploid wheat. Genome Biol. 2023; 24 :7

[171] Zhu X, Xu Z, Wang G. et al. Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem. Genome Biol. 2023; 24 :194